The biological clock in the suprachiasmatic nucleus (SCN) of the hypothalamus generates a near 24-h time base that organizes behaviors into circadian rhythms. Prominent among the rhythms governed by the SCN is the daily cycle of sleep and wakefulness. Cholinergic neurons of the brainstem and basal forebrain, regions that contribute to sleep/arousal states, in turn, project directly to the SCN, providing a route for feedback to the mechanisms that regulate the biological clock in the SCN and the functional contexts in which they act. The PI's extensive studies of the SCN brain slice preparation from rat have established that this circadian clock undergoes spontaneous circadian oscillation in SCN neuronal activity and concomitant modulation of sensitivities to phase-resetting stimuli. By monitoring the activity rhythm of the ensemble of SCN neurons under constant condition in vitro, the investigators have demonstrated that the SCN clock regulates its own sensitivity to afferent signals. The circadian pattern of sensitivities correlates with discrete periods in the environmental cycle of day and night. They have found that the circadian clock is specifically sensitive to robust phase advance by muscarinic cholinergic stimulation during subjective night, but not in the day. Further, nocturnal cholinergic stimuli act via an M1-like muscarinic cholinergic receptor to activate cellular cGMP pathways that advance the timekeeping mechanism. Yhe investigoators propose to use electrophysiological, biochemical and immunocytochemical techniques to probe the mechanism by which acetylcholine regulates the circadian clock at night. Specific aims include: 1) To elucidate the elements of the signal transduction cascade by which nocturnal cholinergic signals reset the SCN timekeeping mechanism; 2) To evaluate transcriptional activation mediated via this pathway; and 3) To localize the cellular constituents and sites of change. These experiments will provide new insights into central muscarinic mechanisms, the effects of cGMP/PKG activation on cell state, and interactions between brain sites regulating sleep/wakefulness and the biological clock. This research has basic relevance to understanding cellular and molecular substrates of both circadian rhythms and sleep. It has applied relevance for developing strategies for drug chronotherapeutics and for ameliorating internal desynchronization manifested as disordered sleep and hormonal patterns, depressive affective disorders, and cognitive impairment (e.g., SDAT) due to decline of the cholinergic system with aging.